Generation of cell polarity is a key biological process that defines multiple aspects of normal cell growth, and differentiation. The major goal of this proposal is to understand how microtubules and microtubule motors contribute to generation of cell polarity and organization of the cytoskeleton. During the previous funding period our group discovered that microtubules in the cell are actively transported in the cytoplasm, with one microtubule serving as a track and another as a cargo for a motor protein. This movement is powered by conventional kinesin (kinesin-1), a major microtubule motor that has previously only been implicated in movement of organelles along microtubules. Microtubule movement by kinesin is required for formation of processes by neurons and non-neuronal cells. Here we propose to study the role of microtubule movements by kinesin and other microtubule motors in generation of cell polarity and specifically in formation of processes by neuronal and non-neuronal cells. We will initially use Drosophila S2 cells for identification of molecular components involved in microtubule sliding and formation of processes because these cells are highly sensitive to protein knock-down by RNAi and because microtubule movement and process formation can easily be visualized, tracked, quantified and manipulated. Once the components are identified, we will use Drosophila neurons to study how these components contribute to formation of neurites in culture and neurogenesis in vivo. We will use tools of Drosophila genetics combined with imaging of live neurons and micromanipulation to address these questions. The three overlapping areas to be investigated in this project are: (i) How does motor-driven microtubule sliding organize polarized microtubule arrays? (ii) How is motor-driven microtubule sliding regulated in a cell? (iii) What is the role of microtubule sliding in axon regeneration after injury? We believe that proteins driving and regulating microtubule sliding in neurons could serve as excellent new therapeutic targets for pharmacological stimulation of neurite outgrowth required for successful treatment of neuronal injuries and neurodegenerative diseases.